Electrochemical Studies of Biological Systems - ACS Publications

1 Spectroelectrochemical

Investigation

of

Vitamin

Β

12

and Related Cobalamins HARRY B. MARK, JR., and THOMAS M. KENYHERCZ Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221

Downloaded by 80.82.77.83 on March 20, 2017 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0038.ch001

PETER T. KISSINGER Purdue University, W. Lafayette, Ind. 47907

This paper discusses three aspects of the spectroelectro chemical study of Vitamin B and related cobalamins: (i) the time resolved spectral study of the autooxidation of cob(I)ala mins u n d e r various experimental conditions; (ii) the electro chemical behavior of the cob(III)alamins, a n d (iii) the electro chemical behavior of 5'-deoxyadenosylcobalamin. Several pre viously unknown features concerning the redox chemistry of these unusual but important complexes are reported. 12

The

Autooxidation

of

B s Under Various Conditions 12

Recent spectroelectrochemical investigations of the oxida tion of cob(I)alamins to cob(III)alamins in various media has yielded the previously unobserved sequence of intermediates and steps involved in the mechanism of this biologically important reaction. This sequence of intermediates disagrees with pre viously speculated mechanisms . The time resolved visible-UV spectra for the reoxidation of the electroreduced Vitamin B type compounds: cyanocobalamin (B ), aquocobalamin (B ) and dicyanocobalamin (B -CN) are reported below. The autooxidation of the cob(I)alamins were carried out under both air and inert argon atmospheres, the electrochemical reoxidation was also studied under these conditions. Mechanisms consistent with the qualitative kinetic data obtained from time resolved spectra are presented. The r e c e n t l y d e v e l o p e d m e r c u r y c o a t e d n i c k e l m i n i g r i d s y s t e m was e m p l o y e d i n a t h i n l a y e r e l e c t r o l y s i s c e l l a s a n o p t i c a l l y t r a n s p a r e n t e l e c t r o d e , Hg-Ni 0TTLE. L L = U T h e Hg-Ni 0TTLE c e l l was m o u n t e d i n a c o m p u t e r i z e d H a r r i c k r a p i d s c a n n i n g d u a l t e a m spectrophotometer,ϋ w h i l e a l l experimental p r o c e d u r e s , i n t e r f a c e design, e l e c t r o c h e m i c a l i n s t r u m e n t a t i o n and computer programming i n c l u d i n g data a c q u i s i t i o n , p r o c e s s i n g a n d r e d u c t i o n have been described p r e v i o u s l y . ^ A l l s o l u t i o n s (except that noted i n F i g u r e 2 ) w e r e ImM i n c o b a l a m i n a n d 1.0M i n N a S 0 i * a s t h e s u p porting e l e c t r o l y t e (the solutions f o r the Bi -CN experiments 1

2

2-8

12

12

12a

12

2

2

1

Sawyer; Electrochemical Studies of Biological Systems ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


2

ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS

w e r e a l s o 0.1M i n N a C N ) . T h e s o l u t i o n s w e r e i n t r o d u c e d i n t o t h e OTTLE c e l l w i t h c o b a l t i n t h e +3 v a l e n c e s t a t e . T h e s o l u t i o n s were t h e n p o t e n t i o s t a t e d a t -1.0 v o l t s v s . SCE u n t i l t h e s p e c t r a changed completely t o t h a t o f t h e Co(I) species » ( s e et h e f i r s t spectrum o f F i g u r e 1) and remained c o n s t a n t ( f o r a p p r o x i mately o n e - h a l f h o u r ) . The c o b ( I ) a l a m i n s o l u t i o n s were a l l o w e d to undergo a u t o o x i d a t i o n i n e i t h e r t h e presence o f a i r d i f f u s i n g i n t o t h e OTTLE c e l l o r u n d e r a n i n e r t a r g o n a t m o s p h e r e . T h e p o t e n t i o s t a t was d i s c o n n e c t e d d u r i n g t h e s e a u t o c a t a l y s i s e x p e r i m e n t s . D u r i n g e l e c t r o r e o x i d a t i o n s t e p - w i s e ( 5 0 mv i n c r e m e n t ) p o t e n t i o s t a t i c p o l a r i z a t i o n ( p o t e n t i o s t a t e d a t each p o t e n t i a l u n t i l s p e c t r a ceased changing) under an i n e r t argon atmosphere was e m p l o y e d . C u r v e A o f F i g u r e 2 shows t h e t y p i c a l t r a n s i e n t s p e c t r u m o f a p a r t i a l l y a i r reoxidized s o l u t i o n o fcob(I)alamin while curve Β i s t h e spectrum o f aquocobalamin, t h e f i n a l o x i d a t i o n product.The p e a k s o f i n t e r e s t h e r e , w h i c h c o r r e s p o n d t o i n t e r m e d i a t e s i n t h e o x i d a t i o n s e q u e n c e o c c u r a t 4 1 0 nm a n d 4 7 5 nm. D u r i n g t h e large scale preparation o fB i t was o b s e r v e d t h a t t h e r e l a t i v e r a t i o o f t h e 4 1 0 a n d 4 7 5 nm p e a k s v a r i e d m a r k e d l y depending on t h e r a t e a t w h i c h o x y g e n was i n t r o d u c e d i n t o t h e c o b ( I ) a l a m i n s o l u t i o n . F o r example, curve A o f F i g u r e 2 i s t h e s p e c t r a ob t a i n e d on bubbling oxygen through a r a p i d l y s t i r r e d B solu t i o n ( t h e 4 1 0 nm p e a k i s much l a r g e r t h a n t h e 4 7 5 nm p e a k ) . How e v e r , t h e 4 7 5 nm p e a k s i n t h e c u r v e s shown i n F i g u r e 3 a r e l a r g e r t h a n t h e 4 1 0 nm p e a k s . Inthe l a t t e r case, a i r d i f f u s e s i n t o the B s o l u t i o n s l o w l y f r o m t h e e d g e s o f t h e OTTLE c e l l . Thus i t i s f e l t t h a t t h e 4 1 0 a n d 4 7 5 nm p e a k s r e p r e s e n t t w o d i f f e r e n t s p e c i e s even though p r e v i o u s workers have r e p o r t e d both peaks a s being c h a r a c t e r i s t i c o fthe s o - c a l l e d B ! The s p e c t r a o b t a i n e d f o l l o w i n g t h e e x h a u s t i v e r e d u c t i o n o f B i , B , a n d B - C N a t - 1 . 0 v o l t s v s . SCE w e r e i d e n t i c a l c o b (I)alamin species corresponding t othose p r e v i o u s l y designated B s . Though t h e e x a c t c o o r d i n a t i o n geometry f o r c o b ( I ) a l a m i n i s unknown, i t has been s u g g e s t e d t h a t t h e b e n z i m i d a z o l e i s i n a base-off c o n f i g u r a t i o n — w i t h water molecules occupying each of the axial positions. As B a n d B appear t o r e o x i d i z e a t comparable r a t e s and have s i m i l a r time r e s o l v e d s p e c t r a l c h a r a c t e r i s t i c s , a l l argu m e n t s made f o r B a r e e q u a l l y a p p l i c a b l e t o B i . A l s o , i t w a s found t h a t t h e time r e s o l v e d s p e c t r a l sequences and r a t e s o f peak c h a n g e s w e r e v i r t u a l l y t h e same i n t h e p r e s e n c e o f a i r o r a r g o n . The t i m e r e s o l v e d s p e c t r a f o r t h e a i r r e o x i d a t i o n o f a c o b ( I ) alamin s o l u t i o n obtained by the exhaustive reduction o f cyanoc o b ( I I I ) a l a m i n i s shown i n F i g u r e s 1 a n d 3. F i g u r e 1 s h o w s t h a t t h e c o b ( I ) a l a m i n , a s m o n i t o r e d b y t h e 3 8 5 nm p e a k , i s v i r t u a l l y r e o x i d i z e d c o m p l e t e l y t o a c o b ( I I ) a l a m i n i n t h e f i r s t 100 seconds. The p e a k w h i c h d e v e l o p s a t 4 7 5 nm c o r r e s p o n d s t o a c o b ( I I ) a l a m i n , — ( d e s i g n a t e d h e r e a s B ) g r o w s t o a maximum i n t h e f i r s t 400 s e c o n d s and t h e n s l o w l y d e c r e a s e s f i n a l l y v a n i s h i n g a t a b o u t i 2 a

1 2 s

1 2 S

i 2 r

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1 2 a

i 2

i 2

i 2

i 2 a

i 2

2 a

i 2 r


Vitamin


MARK E T AL.

o.o Ll 280

I

1

320

360

1

B

12

1

and Related

I

400 440 480 WAVELENGTH . Ν M

Cobalamins

1

1

*-

520

560

600

ο·β

Figure 1. Time-resolved spectra for the reoxidation of cob(I)alamin to cob(II)ahmin in 1.0M lSla S0 at pH = 7.0 in 0-400 sec 2

300

350

400

ll

450 WAVELENGTH

500

550

600

650

, NM

Figure 2. Spectra of the air-reoxidized product formed from the electroreduction of the cyanocob(IU)alamin in 0.1M NaN0 . A, partially reoxidized cob(II)ahmin; B, totally reoxidized cob(IH)alamin, aquocob(III)alamin. 3


4


1

5 χ 10 * s e c o n d s . T h e c y a n o c o b ( I I I ) a l a m i n p e a k o f 3 6 0 nm w h i c h b e g i n s t o d e v e l o p a t 10 s e c o n d s i s c o m p l e t e l y r e c o n v e r t e d t o Bi2 b y 1 0 s e c o n d s ( a s s e e n i n F i g u r e 3 ) . T h e p r e v i o u s l y u n r e p o r t e d i n t e r m e d i a t e e x h i b i t s a 4 1 0 nm p e a k , w h i c h o c c u r s i n t h e same q u a l i t a t i v e t i m e p e r i o d o f t h e c h a r a c t e r i s t i c 4 7 5 nm c o b ( I I ) a l a m i n p e a k , a p p e a r s i n 2 0 0 s e c o n d s , r e a c h e s a maximum b y 5 χ 1 0 s e c o n d s a n d h a s d i s a p p e a r e d b y a b o u t 2 χ 10 s e c o n d s . As t h e r e a r e n o p e a k s i n t h e r e g i o n o f 3 7 0 nm w h i c h w o u l d i d e n t i f y e i t h e r Co(I) o r C o ( I I I ) s p e c i e s ^ during t h i s time i n t e r v a l , i t i s f e l t t h a t t h e p r o n o u n c e d 4 1 0 nm p e a k i n d i c a t e s a s e c o n d , d i f f e r e n t c o b ( I I ) a l a m i n i n t e r m e d i a t e which w i l l be i d e n t i f i e d as Bi2r- A s m e n t i o n e d p r e v i o u s l y , t h e t i m e r e s o l v e d s p e c t r a f o r t h e a i r r e o x i d a t i o n o f cob(I)alamin obtained by t h e exhaus t i v e r e d u c t i o n o f a q u o c o b ( I I I ) a l a m i n , i s q u a l i t a t i v e l y t h e same a s r e p r e s e n t e d i n F i g u r e s 1 a n d 3. H o w e v e r , t h e a i r r e o x i d a t i o n o f c o b ( I ) a l a m i n o b t a i n e d upon e x h a u s t i v e r e d u c t i o n o f d i c y a n o c o b ( I I I ) a l a m i n ( i n t h e p r e s e n c e o f 1 0 0 - f o l d e x c e s s o f CN~) i s q u i t e d i f f e r e n t a s shown i n F i g u r e 4. F i r s t o f a l l , d i c y a n o c o b ( I I I ) alamin i s t o t a l l y regenerated i n l e s s than 400 seconds. The i n c r e a s e / d e c r e a s e i n t h e 2 9 0 nm b a n d , t h e r i s e a n d f a l l o f t h e 475 nm ( c o b ( I I ) a l a m i n ) p e a k , t h e f i n a l r i s e o f t h e 3 6 8 nm p e a k p l u s t h e t o t a l l a c k o f a 4 1 0 nm p e a k i n d i c a t e s t h a t t h e r e o x i d a t i o n o f c o b ( I ) a l a m i n i n t h e presence o f excess c y a n i d e goes through only a c o b ( I I ) a l a m i n , B type i n t e r m e d i a t e . Electrochemical r e o x i d a t i o n o f cob(I)alamin, obtained by the e l e c t r o r e d u c t i o n o f B12 u n d e r a n a r g o n a t m o s p h e r e , g o e s t h r o u g h b o t h t h e B i 2 r ( 4 7 5 nm) a n d B i ( 4 1 0 a n d 4 7 5 nm) i n t e r m e d i a t e s i n t h e p o t e n t i a l r e g i o n f r o m - 0 . 6 0 t o -0.01 v o l t s v s . SCE y i e l d i n g B12 a t +0.10 v o l t s v s . S C E . T h e c o n d i t i o n s o f t h e e l e c t r o c h e m i c a l r e o x i d a t i o n e x p e r i m e n t s i n d i c a t e t h a t t h e 4 1 0 nm p e a k i s not i n d i c a t i v e o f an oxygen adduct type o f cobalamin s p e c i e s . The t i m e r e s o l v e d a u t o o x i d a t i o n s p e c t r a o f a c o b ( I ) i n i m i d e — (no b e n z i m i d a z o l e m o i e t y o n t h e c o r r i n r i n g s y s t e m ) w a s a l s o examined. The a b s o r b a n c e - p o t e n t i a l r e d u c t i o n c h a r a c t e r i s t i c s o f the c y a n o a q u o c o b ( I I I ) i n i m i d e — s t a r t i n g m a t e r i a l and t h e r a t e o f autooxidation a r e p a r a l l e l t o those o f Vitamin B itself indi c a t i n g t h a t t h e l a c k o f t h e b e n z i m i d a z o l e moiety has n o t appre c i a b l y a l t e r e d t h e redox p r o p e r t i e s o f t h e c e n t r a l c o b a l t i o n . However, t h e time r e s o l v e d a u t o o x i d a t i o n s p e c t r a o f t h e c o b ( I ) i n i m i d e d o n o t e x h i b i t a 4 1 0 nm p e a k . As v a r i a t i o n o f p e a k s i n t h e 4 0 0 - 5 0 0 nm r e g i o n h a v e p r e v i o u s l y been a s s o c i a t e d w i t h changes i n t h e a x i a l l i g a n d s , — i t i s a t t r a c t i v e t o s p e c u l a t e a t t h e p o i n t t h a t t h e two c o b ( I I ) a l a m i n s represent c o n f i g u r a t i o n s where t h e benzimidazole i s e i t h e r c o o r d i n a t e d t o t h e C o ( I I ) , a base-on form, o r where t h e benzimi d a z o l e has been r e p l a c e d by a w a t e r i n t h e y - a x i a l p o s i t i o n , a b a s e - o f f f o r m . T h o u g h t h e p o s s i b i l i t y e x i s t s t h a t t h e r e i s some a l t e r a t i o n i n t h e c o r r i n r i n g s t r u c t u r e could a l s o account f o r the observed behavior, i t i s f e l t t h a t redox changes i n t h e r i n g would be i r r e v e r s i b l e . The f a c t t h a t t h e c o b i n i m i d e and k

5


3

h

1

2

r

2 r

i 2


Vitamin

MARK E T AL.

B

îg

and Related

Cobalamins


5.0

0.0

I

1 1 I I I I I I \L 2 . · 2 * > 3 2 O M 0 4 0 0 4 4 O 4 t 0 5 2 O M 0 t 0 0 WAVELENGTH. NM

Figure 3. Time-resolved spectra from the reoxidation of cob(H)ahmin to a cob(III)alamin in 1.0M Na SO at pH = 7.0 in 630-70,000 sec 2

0.0

I

I

280

I

I

320

360

I

1

1

400 440 4*0 WAVELENGTH . NM

Il

1

1

520

560

*- 0.6 600

Figure 4. Time-resolved spectra for the reoxidation of cob(I)alamin to dicyanocob(Ul)alamin in 1.0M Na SO^ and 0.1M NaCN at pH = 11.0 in 0-410 sec 2


6

ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS

d i c y a n o c o b a l a m i n e x p e r i m e n t s show n o 4 1 0 nm b a n d i s c o n s i s t e n t with the base-on/base-off suggestion. Furthermore, i f t h i s i s c o r r e c t , then t h e B l r would be t h e base-on c o n f i g u r a t i o n and B r would be t h e b a s e - o f f form. Q u a l i t a t i v e l y , t h e time resolved spectra i n d i c a t e that t h e reoxidation o f cob(I)alamin i n the presence o fstoichiometric o r l e s s a m o u n t s o f CN~ f o l l o w s t h e r e a c t i o n s c h e m e i l l u s t r a t e d below. As p r e v i o u s l y m e n t i o n e d , t h e p r e s e n c e o f e x c e s s c y a n i d e i o n s a f f e c t s t h e r e o x i d a t i o n scheme s u c h t h a t e i t h e r t h e B J species i s n o t formed o r t h a t t h e o x i d a t i o n o f t h e B species i s kineti c a l l y favored. I t has been p o s t u l a t e d by o t h e r s — t h a t c o b ( I ) alamin autooxidizes t o cob(II)alamin with t h e e v o l u t i o n o f hydro gen a n d t h a t c o b ( I I ) a l a m i n s d i s p r o p o r t i o n a t e i n t h e m e c h a n i s m t o form cob(I) and c o b ( I I I ) alamin s p e c i e s . Under present e x p e r i mental c o n d i t i o n s t h i s d i s p r o p o r t i o n a t i o n mechanism o f c o b ( I I ) alamin i s not d i r e c t l y observed. This i s i n agreement w i t h t h e c o n c l u s i o n s o f B i r k e e t a l Λ who e s t i m a t e d t h a t t h e t h e r m o d y namic and k i n e t i c parameters f o r such a d i s p r o p o r t i o n a t i o n a r e very unfavorable. However, t h e time r e s o l v e d s p e c t r a l sequence observed does n o t r u l e o u t t h e p o s s i b i l i t y t h a t B undergoes disproportionation. I f the rate o fdisproportionation o f B i s very slow compared t o t h e r a t e o f o x i d a t i o n o f B i t h e same time r e s o l v e d s p e c t r a would be o b t a i n e d . I t i s interesting to n o t e t h a t we d o n o t f i n d a n y d i r e c t e v i d e n c e f o r H e v o l u t i o n w h i c h i s e a s i l y o b s e r v e d ( t r a p p e d s m a l l b u b b l e s ) i n t h e OTTLE c e l l f o r s y s t e m s w h e r e i t o c c u r s . We h a v e b e e n u n a b l e t o i d e n t i f y t h e o x i d i z i n g agent(s) thus f a r . F u r t h e r m o r e , i t has n o t been p o s s i b l e t o c a l c u l a t e m e a n i n g f u l k i n e t i c p a r a m e t e r s f r o m the time and p o t e n t i a l r e s o l v e d s p e c t r a a s no q u a n t i t a t i v e d i f f u s i o n model has been p o s t u l a t e d . T h e r e f o r e , c a l c u l a t i o n s o f d i f f u s i o n i n t h e OTTLE t y p e c e l l c a n n o t b e made. F u r t h e r m o r e , we c a n n o t t e l l i f B i oxidizes directly t o both B and B J r a t d i f f e r e n t r a t e s o r t h a t i f B J results simply from a r a p i d e q u i l i b r i u m with B as i l l u s t r a t e d by the d o t t e d a r r o w i n t h e p r o p o s e d m e c h a n i s m u ( C o b a l t +2 c o m p l e x o f t h i s t y p e a r e a l w a y s l a b i l e . — ) T h e same a r g u m e n t a p p l i e s t o t h e interpretation o fB and B ' o x i d i z i n g t o c o b ( I I I ) a l a m i n . However, i t does appear t h a t t h e r e o x i d a t i o n o f B t o dicyanoc o b ( I I I ) a l a m i n o c c u r s much m o r e r a p i d l y t h a n t h e r e o x i d a t i o n o f either B or B l t o B o r B . D i f f u s i o n s t u d i e s a r e now i n p r o g r e s s a s w e l l a s a s i m i l a r s t u d y w i t h m e t h y l a n d 5'-deoxyadenosylcob(III)alamin. Q u a n t i t a t i v e studies o f the chemical and e l e c t r o c h e m i c a l o x i d a t i o n k i n e t i c s a n d m e c h a n i s m s w i l l b e reported a t a f u t u r e date. 2

i 2

2 r


i 2 r

1

2

r

i 2 r

2 S

2

2 S

i 2 r

2

2 r

i 2 r

i 2 r

i 2 r

1

1

The

2

r

2 r

Electrochemical

1 2

Behavior

2

r

i 2 a

of Cob(III)alamins

The e l e c t r o c h e m i c a l b e h a v i o r o f v i t a m i n B (cyanocob(III)a l a m i n ) and r e l a t e d c o b a l a m i n compounds i n aqueous media i s o f i 2


1.

MARK E T A L .

Vitamin

B

12

and Related

7

Cobalamins

importance f o r e l u c i d a t i n g t h e biomechanistic r e a c t i o n sequences which i n v o l v e cobalamin species. There has been c o n s i d e r a b l e study o f the redox processes o f cobalamins using the convention al e l e c t r o a n a l y t i c a ltechniques o f p o l a r o q r a p h y , * " coulom e t r y , * * * and c y c l i c v o l t a m m e t r y , * _ and d i v e r s e working e l e c t r o d e m a t e r i a l s such a s m e r c u r y * ' ~ and p l a t i n u m . i * H o w e v e r , t h e i n t e r p r e t a t i o n o f t h e e l e c t r o c h e m i c a l d a t a t o unam b i g u o u s l y determine even t h e most fundamental parameters such a s t h e t h e r m o d y n a m i c r e d o x p o t e n t i a l s , t h e n u m b e r o f e l e c t r o n s (nvalues) i n v o l v e d i n t h e e l e c t r o n t r a n s f e r s t e p s , and t h e sequence o f s t e p s i n t h e mechanism has n o t been p o s s i b l e because o f numer ous c o m p l i c a t i n g c o n d i t i o n s . The c o m p l i c a t i o n s encompass s t r o n g a d s o r p t i o n o f both r e a c t a n t and product, i r r e v e r s i b i l i t y o f t h e r e d o x r e a c t i o n s , unusual medium e f f e c t s i n v o l v i n g t h e s o l v e n t system and t h e s u p p o r t i n g e l e c t r o l y t e , and marked v a r i a t i o n o f e l e c t r o d e k i n e t i c s with t h e e l e c t r o d e material, «» ~ Recently new t e c h n i q u e s e m p l o y i n g m i n i g r i d e l e c t r o d e s i n c o n j u n c t i o n w i t h t h i n l a y e r e l e c t r o l y s i s c e l l s — h a v e been d e v e l o p e d w h i c h have proved u s e f u l t o t h e study o f t h e b a s i c redox p r o p e r t i e s o f c y t o chrome c . - ^ This paper r e p o r t s t h e r e s u l t s obtained by using t h i n l a y e r m i n i g r i d electrode c e l l s t o study the electrochemical and s p e c t r o e l e c t r o c h e m i c a l b e h a v i o r o f c y a n o c o b a l a m i n ( B ) , aquocobalamin ( B i ) , and dicyanocobalamin (Bi -CN). 9

3

9

3 1

8


8

2 2

3 3

3 5

9

2 2

3 7

3 2

9

2 2

3

3 3

9

1 2

2 a

2

S p e c t r o e l e c t r o c h e m i s t r y o f t h e Cobalamin Systems. As changes i n t h e valence o f c o b a l t , t h e c e n t r a l metal i o n o f t h e cobalamins, a r e r e f l e c t e d by d i s t i n c t changes i n t h e v i s i b l e ab s o r p t i o n s p e c t r a , a c o u p l i n g o f e l e c t r o c h e m i c a l and s p e c t r o s c o p i c m e a s u r e m e n t s was p e r f o r m e d t o e l u c i d a t e t h e r e d o x b e h a v i o r o f t h e cobalamins. S p e c t r o e l e c t r o c h e m i c a l experiments were c a r r i e d o u t u s i n g t h e o p t i c a l l y t r a n s p a r e n t t h i n layer e l e c t r o c h e m i c a l c e l l s (OTTLE) i n t h e presence and a b s e n c e o f t h e e l e c t r o n t r a n s f e r mediator, 2 , 6 - d i c h l o r o p h e n o l i n d o p h e n o l T h e cobalamin-containi n g OTTLE c e l l s w e r e p o t e n t i o s t a t e d w h i l e t h e o p t i c a l a b s o r b a n c e o f a peak o f i n t e r e s t and c u r r e n t l e v e l s were monitored. When both t h e a b s o r b a n c e stopped changing and t h e c u r r e n t l e v e l s had f a l l e n t o e s s e n t i a l l y z e r o (

SCE

Figure 6. Potential-absorbance curves for the reduction (A) and oxidation (B) of 1.2 mM B monitored at 550 nm. 1.0 M Na SO^; 0.1 Μ NaN0 ; pH 7.0; Hg-Ni minigrid; cell thickness, 0.017 cm. 12

2

3




J

I

Ο

I

I

-0.2

I

1

1

-0.4

Potential . Volts

1

1

-0.6 vs

1

-0.·

Ι -10

1

SCE

Figure 7. Potential-absorbance curves for the re duction (A) and oxidation (B) of 1.2 mM Β i2 momtored at 475 nm. 1.0 M Na SO>; 0.1 M NaNO ; pH 7.0; Hg-Ni minigrid; cell thickness, 0.017 cm. 2

Potential

s

Volts

vs

SCE

Figure 8. Potential-absorbance curves for the reduction (A) and oxidation (B) of 1.2 mM B CN monitored at 580 nm. 1.0 M Na S0 ; O.J M KCN; pH 10.4; Hg-Ni minigrid; cell thickness, 0.017 cm. 12

2

4


1.

MARK E T A L .

Vitamin

B

and Related

12

11

Cobalamins

p o t e n t i a l s f o r B12-CN r e d u c t i o n a n d r e o x i d a t i o n c u r v e s i n F i g u r e s 8 and 9 ) . Vitamin B was a l s o i n v e s t i g a t e d a t a H g - N i e l e c t r o d e i n a s i m i l a r set of experiments. C u r v e s A o f F i g u r e 1 0 ( t h e 530-nm p e a k ) a n d F i g u r e 11 ( t h e 475-nm p e a k ) show t h a t B undergoes an u n u s u a l t w o - s t e p p r o c e s s , a s i l l u s t r a t e d b y t h e b r e a k s a t a b o u t -0.06 and -0.65 V , b e f o r e c o m p l e t e c o n v e r s i o n t o a c o b ( I I ) a l a m i n s p e c i e s . C u r v e A o f F i g u r e s 1 0 a n d 11 shows t h a t t h e c o b ( I l ) a l a m i n i s then reduced to cob(I)alamin a s the p o t e n t i a l i n c r e a s e s f r o m -0.8 t o -1.0 V. On r e o x i d a t i o n , c u r v e Β o f F i g u r e 1 1 , t h e c o b ( I ) a l a m i n i s reversibly reoxidized to cob(II)alamin o v e r t h e same p o t e n t i a l r a n g e a s i n t h e negative scan. H o w e v e r , t h e Β c u r v e s i n b o t h F i g u r e s 1 0 and 11 i n d i c a t e t h a t t h e r e o x i dation which corresponds to a q u a n t i t a t i v e regeneration of B i a from the c o b ( I I ) a l a m i n s p e c i e s i s a s i n g l e step process which o d d l y o c c u r s a t a p o t e n t i a l negative t o t h e f i r s t r e d u c t i o n s t e p ( c a . -50 mV).u To c h e c k t h e u n i q u e s p e c t r o e l e c t r o c h e m i c a l p r o p e r t i e s o f B , other samples of B from d i f f e r e n t s o u r c e s and p r e p a r a t i o n s were examined, and, a l s o , the e l e c t r o c h e m i c a l p r e p a r a t i o n was r e c y c l e d a n u m b e r o f t i m e s . The s p e c t r o e l e c t r o c h e m i c a l b e havior at a p a r t i c u l a r wavelength f o r B from the v a r i o u s pre p a r a t i o n s gave s p e c t r o p o t e n t i o s t a t i c curves(OTTLEgrams) i d e n t i c a l with those presented herein. A l s o , s p e c t r o p o t e n t i o s t a t i c c y c l i n g of B gave r e p r o d u c i b l e s e t s of c u r v e s . I t i s i n t e r e s t i n g t h a t the a b s o r b a n c e - p o t e n t i a l waves f o r B i n t h e OTTLE e x p e r i m e n t s do n o t c o r r e s p o n d t o a n y p e a k s i n t h e c y c l i c v o l t a m m o g r a m o f B a t t h e same e l e c t r o d e . H o w e v e r , t h e t h r e e absorbance-poten t i a l "waves" f o r the r e d u c t i o n o f B i do c o r r e l a t e reasonably w e l l w i t h t h e t h r e e waves o b s e r v e d i n the p r e v i o u s l y r e p o r t e d polarography of B . > * . To understand the unusual two-step process i n the reduction of B t o a c o b ( I I ) a l a m i n s p e c i e s and to determine i f the e l e c t r o d e i t s e l f i s p l a y i n g a r o l e i n the e l e c t r o n t r a n s f e r k i n e t i c s , the mediator 2,6-dichlorophenolindop h e n o l was u s e d i n c o n j u n c t i o n w i t h t h e A u m i n i g r i d e l e c t r o d e . — The m e d i a t o r f u n c t i o n s a s t h e p r i m a r y e l e c t r o n t r a n s f e r a g e n t b e t w e e n t h e e l e c t r o d e and a r e d o x s y s t e m t h a t has v e r y s l o w heterogeneous e l e c t r o n t r a n s f e r r a t e s . Thus, the mediator a c c e l erates the o v e r a l l e l e c t r o c h e m i c a l r e a c t i o n of the system of i n t e r e s t . The c h o i c e o f t h i s m e d i a t o r was d e t e r m i n e d b y t h e po t e n t i a l r e g i o n o f i n t e r e s t i n t h i s c a s e (+0.2 t o -0.2 V v s . S C E ) . The A u m i n i g r i d e l e c t r o d e was u s e d t o e l i m i n a t e t h e p o s s i b i l i t y of o x i d a t i o n of the working e l e c t r o d e m a t e r i a l i n t h i s p o t e n t i a l r e g i o n and b e c a u s e t h e c y c l i c v o l t a m m o g r a m s o f B exhibited a m o r e w e l l - d e f i n e d wave a t a n i n t e r m e d i a t e p o t e n t i a l , a n d B appeared t o be l e s s s t r o n g l y adsorbed on the Au e l e c t r o d e . The a b s o r b a n c e c h a n g e s o f t h e 525-nm ( B i ) and 475-nm ( B ) b a n d s a s a f u n c t i o n o f t h e a p p l i e d p o t e n t i a l a r e shown i n F i g u r e s 1 2 a n d 1 3 r e s p e c t i v e l y . C u r v e A o f F i g u r e 1 2 s h o w s o n l y one " w a v e " w i t h a h a l f - a b s o r b a n c e p o t e n t i a l o f -0.15 V i n t h e +0.2 t o -0.6 V i 2 a

1

2

a


2

i 2 a

i 2 a

i 2 a

i 2 a

1

2

a

1 2 a

2 a

9

2 6

2

7

i 2 a

1

2

a

i 2 a

i 2 a

2 a

i 2 r




Potential

Volts vs SCE

Figure 9. Potential-absorbance curves for the reduction (A) and oxidation (B) of 1.2 mM B CN monitored at 475 nm. 1.0 M Na S0 ; 0.1 M KCN; pH 10.4; Hg-Ni minigrid; cell thickness, 0.017 cm. 12

2

4

Figure 10. Potential-absorbance curves for the re duction (A) and oxidation (B) of 0.9 mM Β i2a monitored at 530 nm; 1.0 M Na SO,; 0.1 M NaNO ; pH 7.01; Hg-Ni minigrid; cell thickness, 0.017 cm. 2

s


1.

Vitamin

MARK E T A L .

B

12

and Related

13

Cobalamins

2,08


-0.2

-0.4

Potential

.

-0.6

-as

Volts vs SCE

-1.0

Figure 11. Potential-absorbance curves for the reduction (A) and oxidation (B) of 0.9 mM B monitored at 475 nm. 1.0 M Na SO 0.1 M NaNO ; pH 7.0; Hg-Ni minigrid; cell thickness, 0.017 cm. 12a

2

i;

s

Figure 12. Potential-absorbance curves for the reduction (A) and oxi dation (B) of 0.9 mM B monitored at 525 nm. 1.0 M Na SO 0.1 M NaNO ; pH 7.0; Au minigrid and 2,6-dichlorophenolindophenol; cell thickness, 0.021 cm. 12a 2

0 Potential .

-0.2 Volts

vs

i;

s

-0.4 SCE

.13

Λ Ο

I

09

Figure 13. Potential-absorbance curves for the reduction (A) and oxi dation (B) of 0.9 mM B monitored at 475 nm. 1.0 M Na SO>; 0.1 M NaN0 ; pH 7.0; Au minigrid and 2,6-dichlorophenolindophenol; cell thickness, 0.021 cm. 12a 2

,07

3

0.2

0 Potential

-0.2 Volts

-0.4 vt

SCE

-0.6



14

p o t e n t i a l r e g i o n s c a n n e d . From c u r v e Β o f F i g u r e 12 i t c a n b e seen t h a t the produced B i s totally reoxidized t oB a with l i t t l e h y s t e r e s i s ( h a l f - a b s o r b a n c e p o t e n t i a l o f a b o u t -0.09 V f o r t h e r e o x i d a t i o n ) i n t h e p r o c e s s . T h e c h a n g e s i n t h e 475-nm a b s o r b a n c e p e a k ( F i g u r e 13) a g a i n i n d i c a t e o n l y one "wave" f o r t h e g e n e r a t i o n and s u b s e q u e n t r e o x i d a t i o n o f t h e B r w i t h h a l f - a b sorbance p o t e n t i a l s which correspond f a v o r a b l y t o those o f the B " w a v e s " i n F i g u r e 12. T h u s , t h e m e d i a t o r - A u e l e c t r o d e s y s tem r e f l e c t s a m o r e t y p i c a l r e d o x b e h a v i o r a s i t e l i m i n a t e s t h e unusual h y s t e r e s i s e f f e c t where the r e o x i d a t i o n o f B from B occurred a t p o t e n t i a l s negative t o the i n i t i a l reduction process ( s e e F i g u r e s 1 0 and 1 1 ) . H o w e v e r , a n e x a m i n a t i o n o f t h e m a g n i t u d e o f t h e a b s o r b a n c e c h a n g e o f b o t h t h e 5 2 5 - a n d 475-nm p e a k s s h o w s t h a t i t i s e x a c t l y t h e same a s t h a t f o r t h e f i r s t a b s o r bance waves f o r t h e Hg-Ni e l e c t r o d e — no m e d i a t o r system (see F i g u r e s 1 0 and 1 1 ) , i n d i c a t i n g t h a t e v e n w i t h t h e m e d i a t o r t h e B i s o n l y p a r t i a l l y reduced a t the low n e g a t i v e p o t e n t i a l s . The t o t a l s p e c t r u m o f t h e s o l u t i o n p o t e n t i o s t a t e d a t -0.6 V a l s o i n d i c a t e s t h a t part o f the B ( a p p r o x i m a t e l y 35%) i s u n r e a c t e d . The same r e s u l t w a s a l s o o b t a i n e d f r o m t h e η - v a l u e s t u d i e s ( T a b l e I I I ) a t b o t h t h e H g - N i and A u m i n i g r i d e l e c t r o d e s . T h u s , t h e unusual two p o t e n t i a l processes necessary t o t o t a l l y reduce B appear t o be independent o f both working e l e c t r o d e m a t e r i a l a n d mediator p a r t i c i p a t i o n . Neither the spectra f o r B o r B -CN showed any s i g n i f i c a n t r e d u c t i o n e m p l o y i n g Au m i n i g r e d - m e d i a t o r s y s t e m . No s a t i s f a c t o r y m e d i a t o r w i t h t h e n e c e s s a r y o p t i c a l a n d p o t e n t i a l c h a r a c t e r i s t i c s t o e x p l o r e t h e -0.6 t o -1.0 V p o t e n t i a l a b s o r b a n c e b e h a v i o r a t a H g - N i m i n i g r i d e l e c t r o d e has b e e n f o u n d to date. The h a l f - a b s o r b a n c e p o t e n t i a l s f o r t h e c o b a l a m i n s p e c i e s i l l u s t r a t e d i n F i g u r e s 6 through 13 a r e p r e s e n t e d i n T a b l e I . i 2 r

1 2

1 2

1 2 a


1 2 a

1

2

r

1 2 a

i 2 a

i 2 a

i 2

i 2

η-Value Determination. C o n t r o l l e d p o t e n t i a l coulometry with a t h i n l a y e r m i n i g r i d e l e c t r o d e system^°ιM was used t o determine t h e number o f e l e c t r o n s ( η - v a l u e ) f o r v a r i o u s waves found i n t h e c y c l i c voltammograms o f each o f the c o b a l a m i n s . ^ A typical charge vs. time curve f o r B i s shown i n F i g u r e 14. I t w a s n e c e s s a r y t o e x t r a p o l a t e t h e f i n a l s l o p i n g p o r t i o n o f t h e Q-t curve back t o t = 0 t o c o r r e c t f o r edge e f f e c t s i n h e r e n t i n the t h i n l a y e r c e l l s y s t e m . ^ - The m e t h o d f o r c o r r e c t i o n and c a l c u l a t i o n o f η - v a l u e s f o r c h a r g i n g and r e s i d u a l c u r r e n t b y r e p e a t i n g t h e e x p e r i m e n t o n t h e s u p p o r t i n g e l e c t r o l y t e has b e e n d e scribed p r e v i o u s l y . T h e η-values, as well as the i n i t i a l and f i n a l v a l u e s o f t h e a p p l i e d p o t e n t i a l s t e p s , a r e shown i n T a b l e I I . F o r t h e t h r e e c o b ( I I I ) a l a m i n s y s t e m s u s i n g t h e Hg-Ni m i n i g r i d e l e c t r o d e , o n l y one r e d u c t i o n w a v e i s o b s e r v e d i n t h e -1.0 V v s . SCE p o t e n t i a l r e g i o n and t h e η - v a l u e o b t a i n e d i n e a c h c a s e f r o m t h e Q v s . t d a t a w a s e f f e c t i v e l y two ( 2 ) y i e l d i n g a c o b ( I ) a l a m i n p r o d u c t i n each c a s e w h i c h c o n f i r m s p o l a r o g r a p h i c and other p r e v i o u s l y reported r e s u l t s * * " As expected, the 1 2

2

8

9

2 2

3 7


Vitamin

MARK E T A L .

Table I .

B

12

and Related

Cobalamins

Half-Absorbance Potentials*


Monitor^ Working ed electrodes system Cobalamin Reduction Oxidation wavelength (OTTLE) species mV vs. SCE mV vs. SCE (nm) Hg-Ni

B, "

Hg-Ni

B i 2

Hg-Ni Hg-Ni

Bi2-CN*B, -C!N^

Hg-Ni

B,2a^

Hg-Ni

Au Au Au Au

+ + + +

mediator mediator mediator mediator

2

W

C

2

B,2a^

B, " Bi -CN^

-625 (-875) -625 -875 -850 -825 -910 -60 -635 (-825) -75 -634 -880

-180 (-880) -185 -875 -690 -689 -910

— —

— —

2

C

2

2a

475 580 475 530

-188 (-825) 475 -176 -878

525 475

-93 -110

-155 -140

B, ^

550

a

The cobalamin concentration is 1 mM. It should be pointed out that no relationship between the half-absorbance potentials and the reversible potentials for these species exists at this time. Supporting electrolyte = 1.0 M Na2S0 . < Supporting electrob

4

\Λ \Λ \λ

\ 0J

300

Time

Figure 14. Charge-time curve for the application of a potential step from 0.000 to -0.970 to +0.100 V vs. SCE at a Hg-Ni OTTLE. (A) background, 1.0 M Na SO 0.1 M NaNOs. (B) B , 0.6 mM B , 1.0 M Na SO 0.1 M NaN0 . 2

lt

12

2

i>

h

3


16


background breakdown p o t e n t i a l s h i f t s p o s i t i v e on t h e Au m i n i g r i d e l e c t r o d e a n d o v e r l a p s t h e C o ( I I I ) - C o ( I ) wave o b s e r v e d o n t h e H g - N i e l e c t r o d e . On A u e l e c t r o d e s , B and B i - C N e x h i b i t s a s m a l l prewave a t -0.3 V a n d a broad i r r e v e r s i b l e a p p e a r i n g wave a t a b o u t - 0 . 8 V. T h e B e x h i b i t s a s i n g l e very broad p o o r l y d e f i n e d w a v e w i t h a p e a k p o t e n t i a l a t a b o u t - 0 . 5 V. P o t e n t i a l step experiments w i t h B -CN gave f r a c t i o n a l η-values r e g a r d l e s s of t h e magnitude o f t h e f i r s t a p p l i e d p o t e n t i a l , t h e meaning o f which c o u l d not be i n t e r p r e t e d from t h e e l e c t r o c h e m i c a l data. The η - v a l u e f o r B o n a p o t e n t i a l s t e p t o -0.6 V a l s o y i e l d e d a f r a c t i o n a l v a l u e o f a b o u t 0.65. I t h a s b e e n p r e v i o u s l y r e p o r t e d b y some w o r k e r s t h a t o n l y B can be c o u l o m e t r i c a l l y r e duced t o B ( c o b ( I I ) a l a m i n ) i n a o n e - e l e c t r o n s t e p a t a mer cury electrode a t intermediate p o t e n t i a l s ^ A t a Hg-Ni m i n i g r i d , t h e three c o b ( I I I ) a l a m i n s were c o u l o m e t r i c a l l y reduced t o c o b ( I ) a l a m i n a t - 0 . 9 7 V a n d t h e p o t e n t i a l w a s s t e p p e d t o +0.1 V and t h e Q v s . t c u r v e s w e r e r e c o r d e d . O n l y i n t h e B i c a s e was a r e o x i d a t i o n η-value equal t o 2 found which i n d i c a t e s a v i r t u a l l y q u a n t i t a t i v e r e o x i d a t i o n t o B i . F r a c t i o n a l η-values ob tained f o r B and B i - C N d e r i v e d cob(I)alamins i n d i c a t e s t h a t o n l y p a r t o f these c o b ( I I I ) a l a m i n s a r e regeneraged even a t p o s i t i v e p o t e n t i a l s . However, these r e o x i d a t i o n η-values a r e d i f f i c u l t t o i n t e r p r e t as complicating e f f e c t s a r i s e from the i n t e r f e r i n g m e r c u r y ( I I ) c y a n i d e s p e c i e s w h i c h f o r m i n some c a s e s . A t t h e Au m i n i g r i d e l e c t r o d e s o n l y p a r t o f t h e c o b ( I I I ) a l a m i n s are reduced a s e x p l a i n e d above; however, i t appears from t h e r e o x i d a t i o n η-values t h a t t h e f r a c t i o n reduced i s q u a n t i t a t i v e l y regenerated a tp o s i t i v e p o t e n t i a l s . Thea b i l i t y o f the base-off cobalamin t o form complexes w i t h metal ions a l s o obscures t h e issue.~ F u r t h e r η - v a l u e i n f o r m a t i o n was o b t a i n e d b y f i x e d wave length o p t i c a l monitoring techniques coupled with c o n t r o l l e d p o t e n t i a l coulometry t odetermine η-values f o r appropriate redox processes i n v o l v i n g vitamin B . As mentioned p r e v i o u s l y B was c h o s e n f o r t h i s i n v e s t i g a t i o n a s e a r l i e r s t u d i e s h a d s u g gested that B underwent o n l y a s i n g l e two-electron r e d u c t i o n s t e p . * - . T h e m o n i t o r i n g w a v e l e n g t h o f 4 7 5 nm was c h o s e n a s t h i s peak i s i n d i c a t i v e o f t h e presence ( o r absence) o f a c o b ( I l ) a l a m i n . Monitoring t h i s wavelength, while c o u l o m e t r i c a l l y t h e number o f e l e c t r o n s t r a n s f e r r e d t o t h e c o b a l a m i n i n t h e p r o c e s s i s m e a s u r e d , y i e l d s t h e η - v a l u e f o r each s t e p o f t h e mech anism. Table I I I summarizes t h e r e s u l t s o ft h i s s p e c t r o e l e c t r o chemical study. I t i s e v i d e n t from t h e growth and decay o f t h e 475-nm p e a k t h a t a o n e - e l e c t r o n r e d u c t i o n d o e s o c c u r a t i n t e r mediate p o t e n t i a l s and t h a t t h i s s p e c i e s can undergo a f u r t h e r o n e - e l e c t r o n t r a n s f e r t o form c o b ( I ) a l a m i n . The η - v a l u e i n t h i s case cannot be determined d i r e c t l y because o f i n t e r f e r e n c e from background. This cob(I)alamin i s r e a d i l y r e o x i d i z e d t o a cob( I l ) a l a m i n ; η value equals one. E x i s t i n g experimental c o n d i t i o n s again d i d not allow f o r an accurate determination o f the n-value 1 2

1

2

2

a

1 2

X 2 a

1 2 a

x 2 r


2 6

3 1

2

2

i 2 a

2

i 2

i 2

9

22

33


i 2

1.

MARK E T A L .

Table I I .

Vitamin

B

12

and Related Cobalamins

n- • Value Results

Minigrid working electrode system

Potential step region mV vs. SCE

No. of electrons = η

Species

Reduction Coulometry 0 to -970 B -CN*>

Hg-Ni

1.90 2.00 1.96 0.013 0.126 1.37 0.65

12

B,2a*'

0 to -500 +300 to -400 + 100 to-1000 +300 to -600

Au


17

B,2 a

C

C

e

B -CN -* B -CN > 12

Û

12

B,2a*'

C

Oxidation Coulometry -970 to+100 B,2 ' Bi2-CN '*

Hg-Ni

Û

2.14 0.51 0.38 0.013 0.125 1.40 0.65

C

Û

Bl2a

-500 toO -400 to +300 -1000 to+100 -600 to +300

Au

B,2 ' a

c

Bi2-CN '* fl

B -CN -

a b

X2

Bi2a ' f l

C

a

b

Supporting electrolyte = 1.0 M Na S0 . Supporting electrolyte = 0.1 M KCN. Supporting electrolyte = 0.1 M NaN0 . The cobalamin concentration is 1 mM. 2

4

c

3

Table I I I .

Spectropotential Step Λ-Values for B12

Working electrode system (OTTLE) Hg-Ni

a

Potential step V vs. SCE To From Rest -0.755 Rest -0.755 -1.000 -0.755

-0.755 0.200 -0.755 -1.000 -0.755 0.200

Monitored wavelength No. of electrons (nm) 475 475 475 475 475 475

0.98 a

0.99 0.93 1.04 a

Catalytic process, η > 2.


18


for

the r e o x i d a t i o n to a c o b ( I I I ) a l a m i n .

Conclusions. T h e r e s u l t s d e s c r i b e d a b o v e show t h a t , i n s p i t e o f the f a c t t h a t the e l e c t r o k i n e t i c data are vary compli c a t e d , u n u s u a l , and v i r t u a l l y i m p o s s i b l e t o i n t e r p r e t m e c h a n i s t i c a l l y , the o p t i c a l monitoring o f the s o l u t i o n composition using t h e OTTLE t e c h n i q u e g i v e s a g o o d p i c t u r e o f t h e n e t o r o v e r a l l redox r e a c t i o n s that take place. The f i r s t o b s e r v a t i o n o f s i g n i f i c a n c e i s t h a t a l l t h r e e c o b ( I I I ) a l a m i n s ( B , B12-CN, and B a ) u n d e r g o a q u a n t i t a t i v e o n e - e l e c t r o n r e d u c t i o n t o e i t h e r t h e same o r s i m i l a r c o b ( I I ) a l a m i n ( B i ) s p e c i e s a t i n t e r m e d i a t e p o t e n t i a l s i n t h e 0.0 t o -0.8 V range. Previous electrochemical s t u d i e s by other groups had claimed that only B c o u l d be reduced t o Β at intermediate p o t e n t i a l s . » ~ A s t h e p o l a r o g r a p h i c and c y c l i c v o l t a m m e t r i c s t u d i e s d i d not i n d i c a t e any d i s c e r n i b l e waves i n t h i s p o t e n t i a l r a n g e f o r B o r B - C N , i t a p p e a r s t h a t n o one h a d , t h e r e f o r e , attempted c o u l o m e t r i c r e d u c t i o n s a t such p o t e n t i a l s . However, t h e OTTLE r e s u l t s c l e a r l y show t h a t t h e o n e - e l e c t r o n r e a c t i o n i s common t o a l l t h e s p e c i e s b u t t h a t i n t h e c a s e o f B a n d B - C N the k i n e t i c s o f the r e a c t i o n i s u n u s u a l l y slow even w i t h r e s p e c t t o t h e s l o w s c a n r a t e s e m p l o y e d i n p o l a r o g r a p h y and t h e c y c l i c voltammetry reported here. These one-electron processes f o r B a n d B - C N show u p o n l y d u r i n g p o i n t - b y - p o i n t p o t e n t i o s t a t i c OTTLE t e c h n i q u e s . The r e a s o n f o r t h e e x t r e m e l y s l o w k i n e t i c s o f t h i s o n e - e l e c t r o n r e a c t i o n has n o t b e e n e l u c i d a t e d a t t h i s t i m e . The e l e c t r o n t r a n s f e r r a t e i s f a s t e n o u g h f o r w a v e s t o b e ob served p o l a r o g r a p h i c a l l y or w i t h c y c l i c voltammetry only i n the Β c a s e . U n d e r t h e same c o n d i t i o n s t h e f u r t h e r r e d u c t i o n o f a l l the cobalamin systems from the Co(II) t o Co(I) o x i d a t i o n s t a t e was q u a n t i t a t i v e a n d " r e v e r s i b l e " . The a p p a r e n t h y s t e r e s i s i n v o l v i n g C o ( I I ) to C o ( I I I ) cobalamins i s not p r e s e n t l y w e l l u n d e r s t o o d b u t may r e s u l t f r o m c h e m i c a l r e a c t i o n s i n v o l v e d i n t h e mechanism. It i s i n t e r e s t i n g to note that B -CN i s t o t a l l y re-formed ( s h o w n i n c u r v e B, F i g u r e 9 ) w h i l e c y a n o c o b ( I ) a l a m i n d o e s n o t completely reoxidize to B . T h i s s u g g e s t s t h a t B and B i - C N may r e o x i d i z e b y s e p a r a t e p a t h w a y s . B e c a u s e o f t h e m a g n i t u d e o f the i r r e v e r s i b i l i t y o fthe B r e d o x c o u p l e and a l s o t h e f a c t that B i s n o t t o t a l l y r e - f o r m e d (some B i appears t o be a minor r e o x i d a t i o n product), i t i s thought that on electrochemical reoxidation that B i s t h e i n i t i a l p r o d u c t f o r m e d and t h a t B subsequently forms on a l i g a n d exchange r e a c t i o n i n v o l v i n g the cyanide i n s o l u t i o n ( i n i t i a l l y r e l e a s e d i n t o the s o l u t i o n phase during the reduction o f B t o B i r » a s shown b y t h e f a c t t h a t a -0.1 t o -0.8 V OTTLE e x p e r i m e n t ( c o b ( I I I ) a l a m i n cob(II)alamin) with vitamin B shows t h e same l a r g e i r r e v e r s i b i l i t y i n d i c a t i n g t h a t t h e CN" i s l o s t i n t h e f i r s t r e d u c t i o n s t e p ) . T h i s l i g a n d exchange r e a c t i o n o f B i w i t h C N " has b e e n shown t o b e v e r y f a s t . — However, the net r a t e i s slow because o f the d i l u t e i 2

1 2

2 r

i 2 a


6

2 6

i 2

1

2

Γ

3 1

i 2

1 2

1 2

1 2

1 2

a

1 2

i 2

i 2

2

i 2

1 2

2 a

X 2 a

i 2

i 2

2

i 2

2 a


1.

Vitamin

MARK E T AL.

B

and Related

12

19

Cobalamins

s o l u t i o n s employed. The c o m p l e t e r e g e n e r a t i o n o f B i s not pos s i b l e a s some CN' i s l o s t , p r o b a b l y t h r o u g h t h e f o r m a t i o n o f s t a b l e m e r c u r y ( I I ) cyanide complexes. I t was n o t e d t h a t t h e p e r cent recovery increased on a d d i t i o n of excess cyanide which i s c o n s i s t e n t w i t h t h i s i n t e r p r e t a t i o n . With r e s p e c t to B -CN, the f i n a l p r o d u c t i s formed d i r e c t l y upon r e o x i d a t i o n o r t h e f o l l o w up l i g a n d e x c h a n g e r e a c t i o n b e t w e e n t h e c o n c e n t r a t e d c y a n i d e s o l u t i o n a n d t h e B , f o r m e d b y t h e l o s s o f one e l e c t r o n f r o m B (with water molecules i n the a x i a l p o s i t i o n s ^ - ) , i s very fast. At t h i s time i t i s i m p o s s i b l e t o d i s t i n g u i s h between t h e s e two m e c h a n i s m s f o r t h e r e o x i d a t i o n o f B - C N . H o w e v e r , i t s h o u l d be n o t e d t h a t t h e B s p e c t r a ( a s i n d i c a t e d b y t h e 475-nm p e a k ) i n b o t h B and B - C N r e a c t i o n s a r e v i r t u a l l y i d e n t i c a l . P e r h a p s t h e m o s t u n u s u a l and d i f f i c u l t t o u n d e r s t a n d r e s u l t i s t h e o b s e r v a t i o n o f two d i f f e r e n t p o t e n t i a l - a b s o r b a n c e " w a v e s " f o r the reduction of B t o a c o b ( I I ) a l a m i n . The OTTLE s p e c t r a and t h e a p p a r e n t η - v a l u e d a t a i n d i c a t e t h a t B convertst o B ( a b o u t 65%) a t p o t e n t i a l s a r o u n d -0.05 V a t b o t h t h e H g - N i and Au e l e c t r o d e s w h i l e i t i s n e c e s s a r y t o r a i s e t h e p o t e n t i a l t o g r e a t e r t h a n -0.6 V w h e r e t h e s e c o n d w a v e c o r r e s p o n d i n g t o t h e r e d u c t i o n o f the r e m a i n i n g 35% o f the B i s o b s e r v e d . The m o s t obvious c o n c l u s i o n t h a t f i t s the data q u a l i t a t i v e l y i s t h a t the B e m p l o y e d i n t h e s e e x p e r i m e n t s was i m p u r e a n d c o n t a i n e d a b o u t 35% o f B itself (B was p r e p a r e d f r o m B ) . However, a s p o i n t e d o u t a b o v e , we f o u n d t h a t a l l b a t c h e s o f B gave the same r e s u l t s w h i c h a g a i n w o u l d n o t b e e x p e c t e d t o r e m a i n c o n s t a n t i f the various synthesis routes y i e l d e d only p a r t i a l conversion. A l s o t h e s p e c t r a l and p o l a r o g r a p h i c p r o p e r t i e s d o n o t s u g g e s t t h a t any a p p r e c i a b l e c o n c e n t r a t i o n o f B r e m a i n u n c o n v e r t e d and a l s o a r e i d e n t i c a l w i t h t h e s p e c t r a and p o l a r o g r a p h i c p r o p e r t i e s of vitamin B produced by the t o t a l l y d i f f e r e n t procedures. Furthermore, there i s considerable other i n d i r e c t evidence t h a t t h e r e i s no s i g n i f i c a n t u n c o n v e r t e d B i n the B samples. Note f i r s t o f a l l t h a t t h e r e i s no-0.6 V p o l a r o g r a p h i c wave f o r B t h a t c o r r e s p o n d s t o t h e wave f o r t h i s s e c o n d B species. ( I t i s i n t e r e s t i n g to note t h a t previous p o l a r o g r a p h i c s t u d i e s had r e f e r r e d t o t h e w a v e a t -0.6 V a s a n i m p u r i t y . ) > * * A l though the c y c l i c voltammogram f o r B does e x h i b i t an a n o d i c p e a k a t -0.28 V w h i c h c o u l d b e i n d i c a t i v e o f Hg o x i d a t i o n i n t h e p r e s e n c e o f a c o m p l e x i n g l i g a n d , t h i s w a v e i s a b o u t 5 0 mV p o s i t i v e t o t h e peak c o r r e s p o n d i n g t o m e r c u r y - c y a n i d e f o r m a t i o n i n the B c y c l i c voltammogram and t h e r e i s no c o r r e s p o n d i n g c a t h o d i c sweeps o f B i t s e l f . F i n a l l y high p r e s s u r e l i q u i d chroma t o g r a p h y ( u s i n g a m i x t u r e o f e i t h e r 80% i s o p r o p y l a l c o h o l and 20% w a t e r , o r 65% methanol and 35% w a t e r , a t 2000 p s i o n a n A m i n e x A - 4 c o l u m n , w i t h d e t e c t o r w a v e l e n g t h s e t a t λ 360 nm) o n Bi h a s e x h i b i t e d two c l o s e l y s p a c e d y e t d i s t i n c t p e a k s b o t h w i t h r e t e n t i o n times t h a t are d i f f e r e n t than B . Also, a thin l a y e r c h r o m a t o g r a p h i c c o m p a r i s o n o f B and B u s i n g a 65% 1 2

1 2

1 2 a

1 2 r

1 2

i 2 r


i 2

1 2

1

2

a

1

2

a

1 2 r

1

2

a

1 2 a

i 2

i 2 a

1 2

1

2

a

1 2

1

2

a

1 2

1

1 2

1

2

31

1

2

2

a

a

6

a

1 2

1 2

2 a

1 2

1 2

1

2

a


ELECTROCHEMICAL

20

STUDIES O F BIOLOGICAL

SYSTEMS

m e t h a n o l a n d 3 5 % w a t e r s o l v e n t s y s t e m has shown t h a t B and Bi2a have r e l a t i v e f r o n t s , t h o u g h no s e p a r a t i o n o f B a itself was o b s e r v e d . I t i s f e l t . t h a t r i n g s t r u c t u r e d i f f e r e n c e s would n o t a c c o u n t f o r t h e two u n i q u e B a s p e c i e s , a s t h e B a p r e p a r e d f r o m t h r e e t e c h n i q u e s ( p o t e n t i o s t a t i c , b i o l o g i c a l and c h e m i c a l ) would not g i v e i d e n t i c a l 65/35 r a t i o o f c o n c e n t r a t i o n s . Moreo v e r , i t i s h a r d t o u n d e r s t a n d how two d i f f e r e n t r i n g s , w h i c h w o u l d b e e x p e c t e d t o b e common t o a l l c o b a l a m i n s , e x h i b i t d r a s t i c reduction potential differences for B and n o t f o r B o r B CN. T h u s , i t i s a t t r a c t i v e t o s p e c u l a t e t h a t t h e two s p e c i e s r e present d i f f e r e n c e s i n a x i a l l i g a n d c o n f i g u r a t i o n . The simplest a n s w e r w o u l d b e t h a t one o f t h e B i species c o n t a i n s water molec u l e s i n t h e X and Y p o s i t i o n s ( t h e " b a s e - o f f " f o r m ) w h i l e t h e o t h e r i s i n t h e c o n f i g u r a t i o n w i t h one w a t e r i n t h e X p o s i t i o n and t h e 5 , 6 - d i m e t h y l b e n z i m i d a z o l e i n t h e Y p o s i t i o n ( t h e " b a s e on" f o r m ) . T h e s p e c t r o e l e c t r o c h e m i c a l d a t a c l e a r l y d e m o n s t r a t e t h a t t h e two B s p e c i e s are not i n e q u i l i b r i u m . However, T h u s i u s has shown t h a t t h e X p o s i t i o n o f B i s very l a b i l e ( r a t e c o n s t a n t s o f about 170-2300 M - s " ) . — However, no measurem e n t s h a v e b e e n made o n t h e Y p o s i t i o n b e n z i m i d a z o l e - H 0 e x c h a n g e r a t e s . — I t i s p o s s i b l e t h a t t h i s exchange c o u l d be very slow. The f a c t t h a t t h e d i a q u o c o b ( I I I ) i n a m i d e ( h a v i n g n o b e n z i m i d a z o l e a t t a c h e d t o t h e c o r r i n r i n g s i d e c h a i n ) has b e e n r e p o r t e d t o b e d i f f i c u l t t o r e d u c e ( t f w — -0.7 V ) — i s c o n s i s t e n t b u t n o t p r o o f of the "base-on"-"base-off" explanation. This f a c t suggests that t h e " b a s e - o n " a q u o c o b ( I I I ) a l a m i n f o r m has a c o n f i g u r a t i o n f a v o r a b l e t o r e d u c t i o n ( t h e -0.15 V w a v e ) and t h e " b a s e - o f f " f o r m which would c l o s e l y correspond t o a diaquocob(III)inamide c o n f i g u r a t i o n i s d i f f i c u l t t o r e d u c t (-0.6 V w a v e ) . - ^ H o w e v e r , r e c e n t s p e c t r o e l e c t r o c h e m i c a l s t u d i e s b y L e x a and S a v e a n t ^ - h a v e shown that a tplatinum grid electrodes B does not e x h i b i t t h i s two w a v e o n e - e l e c t r o n b e h a v i o r . O n l y one w a v e i s o b s e r v e d a t a b o u t 0.0 V. F u r t h e r m o r e , t h e y h a v e a l s o shown t h a t t h e d i a q u o c o b ( I l l ) i n a m i d e o x i d i z e s m e r c u r y m e t a l s p o n t a n e o u s l y and f o r m s t h e c o b ( I I ) i n a m i d e . Thus, t h e r e p o r t e d v a l u e s f o r t h i s compound * a r e i n c o r r e c t and r e a l l y c o r r e s p o n d t o t h e s u b s e q u e n t e l e c t r o chemical r e d u c t i o n o f the Cob(II)inamide. Thus, i t appears t h a t t h e m e r c u r y e l e c t r o d e i s i n some w a y t h e c a u s e o f t h i s two w a v e one-electron observation. I t i s o b v i o u s t h a t t h e r e a r e many u n a n s w e r e d q u e s t i o n s c o n c e r n i n g r a t e s o f the m i c r o s c o p i c precesses i n v o l v e d i n the redox c h e m i s t r y o f cobalamin complexes. However, the macroscopic r e sultant e f f e c t o felectrode potential i n s o l u t i o n composition i s now w e l l d e f i n e d . W i t h t h i s b a s i c o v e r a l l m e c h a n i s t i c i n f o r m a t i o n , a m o r e c o m p r e h a n s i v e s t u d y o f t h e e l e c t r o d e k i n e t i c s and time resolved s p e c t r a l s t u d i e s on p o t e n t i a l step experiments on t h e s e a n d o t h e r c o b a l a m i n s u n d e r v a r i a b l e c o n d i t i o n s o f pH, s u p p o r t i n g e l e c t r o l y t e , and e l e c t r o d e m a t e r i a l may e l u c i d a t e a l l t h e steps i n the o v e r a l l mechanism. i 2

i 2

i 2

X 2

1

2

a

i 2

1 2


2 a

i 2 a

i 2 a

1

2

2

1

1

2

a

1


7

1.

Vitamin

MARK E T A L .

B

and Related

12

Cobalamins

21

The E l e c t r o c h e m i c a l B e h a v i o r o f 5 ' - D e o x y a d e n o s y l - C o b a l a m i n enzyme B i T T

(Co-

The u n d e r s t a n d i n g o f t h e r e d o x p r o c e s s e s o f t h e c o b a l a m i n coenzyme, 5'-deoxyadenosylcobalamin (coenzyme B o r A d e n - B ) , is fundamental i n c o n s t r u c t i n g a v a l i d r e a c t i o n sequence f o r cob a l a m i n compounds i n n a t u r e . H i l l O f p a r t i c u l a r i m p o r t a n c e i s t h e o x i d a t i o n s t a t e o f c o b a l t i n A d e n - B and i t s i n f l u e n c e o n the r e a c t i v i t y of the cobalamin species. As a matter of notat i o n a l e a s e and t o m a i n t a i n c o n s i s t e n c y w i t h e x i s t i n g v i t a m i n B c o n c e p t s , t h e c o b a l a m i n c o e n z y m e has b e e n p r i m a r i l y i n t e r preted i n terms of a C o and a n a d e n s y l c a r b a n i o n . — R e d o x mesomers c o n s i s t i n g o f a C o and a n a d e n o s y l r a d i c a l o r a C o and a n a d e n o s y l c a r b o n i u m i o n h a v e a l s o b e e n f o r m a l l y c o n s i d e r e d . A l t h o u g h numerous p u b l i c a t i o n s have assumed the c o b a l t t o be i n t h e +3 o x i d a t i o n s t a t e , * " l i t t l e e v i d e n c e f o r t h i s a s s i g n m e n t i s a v a i l a b l e . Some e v i d e n c e e x i s t s w h i c h s u p p o r t s t h e c l a i m t h a t t h e c o b a l t o f t h e c o b a l a m i n s h o u l d b e c o n s i d e r e d as a d i v a l e n t species.^-Z! S i m i l a r e l e c t r o c h e m i c a l and s p e c t r o e l e c t r o c h e m i c a l t e c h n i ques u t i l i z i n g a n amalgamated g o l d m i n i g r i d e l e c t r o d e i n a t h i n l a y e r c o n f i g u r a t i o n have been employed t o examine t h e r e d o x sequence o f 5'-deoxyadenosylcobalamin. This i n v e s t i g a t i o n suggests t h a t the cobalamin coenzyme undergoes a s i n g l e e l e c t r o n r e d u c t i o n to form v i t a m i n B ç , i n d i c a t i n g cleavage of the c o b a l t - c a r b o n b o n d . The r e o x i d a t i o n o f t h e r e d u c e d s y s t e m c o n t a i n i n g t h e B ^ o c c u r s v i a two c o n s e c u t i v e s i n g l e e l e c t r o n t r a n s f e r s r e s u l t i n g i n the q u a n t i t a t i v e formation of vitamin B , aquocobalamin. High performance l i q u i d chromatography confirmed t h a t 5'-deoxyadenos i n e i s the u l t i m a t e form of the cleaved 5'-deoxyadenosyl moiety. These r e s u l t s i n d i c a t e t h a t 5'-deoxyadenosyl-cobalamin i s reduced by a s i n g l e e l e c t r o n to form the c o b ( I ) a l a m i n , B , w h i c h i s t h e common r e d u c t i o n p r o d u c t o f a l l c o b a l a m i n s p e c i e s . — The f o r m a t i o n o f t h e c o b ( I ) a l a m i n , B , f r o m A d e n - B i s s i g n i f i c a n t as numerous p u b l i c a t i o n s ^ have a l l u d e d t o t h e f a c t t h a t due t o t h e h i g h n u c l e o p h i l i c i t y o f t h e c o b ( I ) a l a m i n s p e c i e s i t may b e t h e b i o l o g i c a l l y a c t i v e f o r m o f t h e c o e n z y m e . The r e s u l t reported herein i s the f i r s t electrochemical evidence f o r the f o r m a t i o n o f the c o b ( I ) a l a m i n from the cobalamin coenzyme. That B can be c o m p l e t e l y r e o x i d i z e d t o B , a c o b ( I I I ) a l a m i n , i s s i g n i f i c a n t i n t h a t e x i s t i n g c y c l i c biochemical mechanisms i n volve Aden-B a s the i n i t i a l l y r e a c t i v e s p e c i e s . ^ Furthermore, r i b o n u c l e o t i d e r e d u c t a s e has shown s p e c i f i c a c t i v i t y t o w a r d B s and A d e n - B i n t h e p r e s e n c e o f ô'-deoxyadenosine.^- -»- -!*1 2

1 2

1 2

1 2

3 +


2 +

1

9

1 +

6 3

i 2

1 2

1 2 a

i 2 S

1 2 S

1 2 S

1 2

X 2 a

1 2

1 2

3

7

1 2

Acknowledgments T h i s r e s e a r c h was s u p p o r t e d i n p a r t b y t h e N a t i o n a l S c i e n c e F o u n d a t i o n , NSF CHE76-04321 and t h e N a t i o n a l I n s t i t u t e s o f H e a l t h , GM-22713-01.


22

electrochemical studies of biological systems

Literature Cited 1. 2. 3. 4. 5. Downloaded by 80.82.77.83 on March 20, 2017 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0038.ch001

6. 7. 8. 9. 10. 11. 12. 13.

14. 15.

16. 17. 18. 19. 20.

Pratt, J. M., "Inorganic Chemistry of Vitamin B ," Academic Press, New York, Ν. Y., 1972, pp. 15-17. Huennekens, F. M., in "Biological Oxidations," Thomas P. Singer, Ed., Interscience Publishers, New York, Ν. Y., pp. 482-502. Tackett, S. L . , Collat, J. W., and Abbot, J. C., Biochemis try, (1963), 2, 919. Collat, J. W. and Abbot, J. C., J. Amer. Chem. Soc., (1964), 86, 2308. Schrauzer, G. N., Deutsch, E. and Windgassen, R. J., J. Amer. Chem. Soc., (1968), 90, 2441. Yamada, R., Shimizu, S. and Fukui, S., Biochemistry, (1968), 2, (7), 1713. Rudiger, H., Eur. J. Biochem., (1971), 21, 264. Birke, R. L . , Brydon, G. Α., and Boyle, M. F., Electroanal. Chem., (1974), 52, 237. Kenyhercz, T. M. and Mark, Jr., Η. B., Anal. Lett., (1974), 7, 1. Heineman, W. R., Norris, B. J. and Goelz, J. Anal. Chem., (1975), 47, 79. Heineman, W. R., DeAngelis, T. P. and Goelz, J., Anal. Chem., (1975), 47, 1364 Kenyhercz, Τ. Μ., DeAngelis, T. P., Norris, B. J., Heineman, W. R. and Mark, Jr., H. B., J. Amer. Chem. Soc., (1975), 98, 2469. However, experiments were performed with the minigrid elec trode area increased to occupy the entire cell volume to determine if the edge effects from the diffusion of unreduced cob(III)alamins from solution not in immediate contact with the minigrid affected the time resolved spectra. No signifi cant difference was observed. Strojek, J. W., Gruver, G. and Kuwana, T., Anal. Chem., (1969), 41, 481. Mark, Jr., H. B., Wilson, R. M., Miller, T. L . , Atkinson, T. V., Yacynych, A. M., and Woods, H., "The On-Line Computer in New Problems in Spectroscopy: Applications to Rapid Scanning Spectroelectrochemical Experiments and Time Resolved Phosphorescence Studies" in "Information Chemistry; Computer Assisted Chemical Research Design," S. Fujiwara and H. B. Mark, Jr., Eds., University of Tokyo Press, Tokyo, Japan, 1975, pp. 3-28. Beaven, G. H. and Johnson, Ε. Α., Nature, (1955), 176, 1264. Ref. 1, p. 184. Ref. 1, p. 20 to 27. Ref. 1, p. 55 The cyanoaquocol(III)inimide was prepared by a previously described method; Ref. 1, p. 294. 12


1.MARKETAL. 21. 22. 23. 24. 25. 26. Downloaded by 80.82.77.83 on March 20, 2017 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0038.ch001

27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

23

Vitamin B and Related Cobalamins 12

Basolo, F. and Pearson, R. G., "Mechanisms of Inorganic Re actions," John Wiley and Sons, Inc., New York, Ν. Y . , pp. 141-144. Diehl, H., Sealock, R. R., and Morrison, J., Iowa State Coll. J. S c i . , (1950), 24, 433. Diehl, H., Morrison, J . I., and R. R. Sealock, Experientia, (1951), 7, 60. Diehl, J., and Morrison, J . I., Rec. Chem. Prog. (1972), 31, 15. Boos, R. N., Carr, J . E . , and Conn, J . B., Science, (1953), 117, 603. Jaselskis, B. and Diehl, H., J. Am. Chem. Soc., (1959), 81, 4345. Jaselskis, B. and Diehl, H . , J . Am. Chem. Soc., (1958), 80, 2147. Collat, J . W., and Tackett, S. L., J . Electroanal. Chem., (1962), 4, 59. Tackett, S. L . , Ph.D. Thesis, Ohio State University, 1962. Kratochvil, B., and Diehl, H., Talanta, (1966), 13, 1013. Hogenkamp, H. P. C. and Holmes, S., Biochemistry (l970), 9, 1888. Lexa, D. and L'hoste, J . M., in "Biological Aspects of Elec trochemistry," G. Milazzo, P. E. Jones, and L. Rampazzo, Ed., Birkhauser Verlag, Stuttgart, 1971, pp. 395-404. Abd-el-Nabey, Β. Α., J . Electroanal. Chem., (1974), 53, 17. Das, P. K. et al., Biochim. Biophys. Acta., (1967), 141, 644. Tackett, S. L. and Ide, J . W., J . Electroanal. Chem. (l971 ), 30, 510. Swetik, P. G., and Brown, D. G., J. Electroanal. Chem., (1974), 51, 433. Kenyhercz, T. M. and Mark, J r . , Η. B., in preparation. Murray, R. W., Heineman, W. R., and O'Dom, G. W., Anal. Chem., (1967), 39, 1666. Provided by Dr. E. A. Deutsch, Department of Chemistry, University of Cincinnati. Beaven, G. H. and Johnson, Ε. Α., Nature (London), (1955), 176, 1264. It should be pointed out the reaction B = [H ] B (hydroxycob(III)alamin) has a pk of 7.8 and a more negative reduction potential than B : H. O. A. H i l l , "Inorganic Biochemistry," Vol. 2, G. Eichcon, Ed., Elsevier, New York, Ν. Y., 1973, Chapter 30. This proton equilibrium is un doubtedly fast and, thus, only the reduction of the B will be observed in these OTTLE experiments. McDuffie, B., Anderson, L. B., and Reilley, C. N., Anal. Chem., (1966), 38, 883. Cotton, F. A. and Wilkinson, G., "Advanced Inorganic Chemis try," 3d ed., Interscience, New York, Ν. Y., 1972, p. 519. Thusius, D. J . Am. Chem. Soc., (1971), 93, 2629. +

+

12a

12b

a

12a

12s

42. 43. 44.


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ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYS Kenyhercz, T. M., Yacynych, Α. Μ., and Mark, J r . , H. B., Anal. Lett., (1976), 9, 203. Reference 6, p. 4346. Reference 11, p. 1889. Lexa, D. and Saveant, J . M., University of Paris, Private Communication, 1976. Babior, Β. M. in "Cobalamin: Biochemistry and Pathophysiol ogy," Β. M. Babior, Ed., John Wiley and Sons, New York, N.Y., 1975, p. 141. Pratt, J . M., "Inorganic Chemistry of Vitamin B12," Academic Press, London, 1972, p. 296. Mahler, H. R. and Cordes, Ε. Η., "Biological Chemistry," Harper and Row, Evanston, Ill., 1971, p. 427. H i l l , J . Α., Pratt, J . M., and Williams, R. J. P., J . Theor. Biol., (1962), 3, 423. Hogenkamp, H. P. C . , Barker, H. A. and Mason, H. S., Arch. Biochem. Biophys., (1963), 100, 353. H i l l , J . Α., Pratt, J . M. and Williams, R. J . P., J . Chem. Soc., (1964), 5149. Pratt, J . M., J . Chem. Soc., (1964), 5154. Huennekens, F. M. in "Biological Oxidations," T. P. Singer, Ed., John Wiley and Sons, New York, Ν. Y . , 1968, p. 483. Hogenkamp, H. P. C. and Holmes, S., Biochem., (1970), 9, 1889. Cotton, F. A. and Wilkinson, G., "Advanced Inorganic Chemis try," John Wiley and Sons, New York, Ν. Y. 1972, p. 888. Hughes, M. N., "The Inorganic Chemistry of Biological Pro cesses," John Wiley and Sons, New York, Ν. Y . , 1974, p. 187. Costa, C . , Puzeddu, A. and Reisenhofer, E . , Bioelectrochem. Bioenerg., (1974), 1, 29. H i l l , H. A. O. in "Inorganic Biochemistry," G. L. Eichhoren, Ed., Elsevier, New York, Ν. Y . , 1975, p. 1076. Babior, Β. M., Acc. Chem. Res., (1975), 8, 378. Abeles, R. H. and Dolphin, D., Acc. Chem. Res., (1976), 9, 114. Seki, H., Shida, T . , and Imamura, Μ., Biochem. Acta., (1974), 372, 106. H i l l , H. A. O., Pratt, J . M. and Williams, R. J . P., Disc. Farad. Soc., (1969), 16S. Kratochvil, B. and Diehl, H., Talanta, (1966), 13, 1013. Nowick, L. and Pawelkiewicz, Bull. Acad. Pol. Sci. C l . II., (1960), 17., 433. Johnson, A. W. and Shaw, N., Proc. Chem. Soc., (1960), 420. Bernhauser, K., Gaiser, P., Muller, O., Muller, E . , and Gunter, F . , Biochem. (1961), 333, 560. Johnson, A. W., Mervyn, L., Shaw, N., and Smith, E. L., J . Chem. Soc., (1963), 4146. White, Α., Handler, P., and Smith, E. L., "Principles of Biochemistry," McGraw H i l l , St. Louis, Mo., 1973, p. 1173. Schrauzer, G. N. and Sibert, J . W., J . Amer. Chem. Soc., (1970), 92, 1022, and references therein.


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Hamilton, Α., Yamada, R., Blakley, R. L., Hogenkamp, H.P.C., Looney, F. D., and Winfield, M. E., Biochem. (1971), 10, 347. Tamao, Y. and Blakley, R. L., Biochem., (1973), 12, 24.


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